Detection of low concentration biological agents

ABSTRACT

Provided are methods of preparing a sample for detection by placing the sample on a shrinkable scaffold and then shrinking the scaffold. An exemplary shrinkable scaffold is a thermoplastic substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional 61/440,350, filed Feb. 7, 2011, the contents of which arehereby incorporated by reference into the present disclosure.

FIELD OF THE INVENTION

The invention disclosed herein related to the field of detection ofbiological samples.

BACKGROUND

The amount of a samples available for clinical diagnostics is typicallylimited or the concentration is low, which makes accurate detectiondifficult. Therefore, there is unmet market need of methods and devicesfor assaying samples with high sensitivity.

SUMMARY

The present disclosure provides methods and devices for measuringsamples, such as biological samples, especially those at low abundance,with high sensitivity and at low cost. In one embodiment, a sample isdisposed on a shrinkable scaffold, and then the shrinkable scaffold isshrunk, reducing the area where the sample is distributed, so as toeffectively concentrate the sample on the surface of the scaffold. Theincrease of concentration can be many fold and lead to greatly increasedsensitivity of detection.

It is further contemplated that, in the event that a biological sample,e.g., protein or nucleic acid, is covalently attached to a scaffoldhaving a silica structure, the great increase in signal enhancement isalso due to the optical effects stemming from covalent linkage of thebiological sample onto the silica structure of the scaffold.

Thus, in one embodiment, the present disclosure provides a method forpreparing a sample for detection, comprising shrinking a thermoplasticmaterial comprising a sample disposed on the thermoplastic material,thereby concentrating the sample on the thermoplastic material.

Likewise, another embodiment provides a method for detecting a samplecomprising (A) disposing the sample on a thermoplastic material, (B)shrinking the thermoplastic material, and (C) detecting the sample.

In one aspect, the sample comprises a detectable label. In anotheraspect, the detectable label comprises a fluorescent agent. In yetanother aspect, the sample is a biological sample.

In some aspects, detecting comprises measuring the amount of a signalemitted by the sample. If the sample comprises a detectable label,detection can be done by measuring emission of the detectable label. Ifthe sample is not labeled, a detectable label can be added so that itbinds to the samples effecting detection of the sample.

The material can be pre-stressed prior to shrinking. When the materialis prestressed, the shrinking can be achieved by removing the stress. Inanother aspect, the shrinking is achieved by heating the material,whether the material has been prestressed.

In some aspects, the shrinking is uniaxial or biaxial. In some aspects,the material is shrunk by at least 60% or more.

Thermoplastic materials suitable for practicing the present technologyinclude, without limitation, a high molecular weight polymer,polyolefin, polyethylene, acrylonitrile butadiene styrene (ABS),acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA),ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP,PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVCalloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal),polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile),polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone(PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutyleneterephthalate (PBT), polyethylene terephthalate (PET), PolycyclohexyleneDimethylene Terephthalate (PCT), polycarbonate (PC),polyhydroxyalkanoates (PHAs), polyketone (PK), polyester polyethylene(PE), polyetheretherketone (PEEK), polyetherimide (PEI),polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC),polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP),polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide(PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU),polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) or spectralon.In one aspect, the thermoplastic material comprises polyolefin. Inanother aspect, the thermoplastic material comprises polyethylene.

Also provided is a substrate having a textured surface prepared by amethod of any one of above embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the immunoassay on the silicananostructures. Bodily fluid such as saliva is deposited on pre-shrunksilica and then incubated with the primary rabbit IgG antibody. Thesubstrate is blocked with BSA before being washed with PBS. Secondarygoat anti-rabbit antibody IgG conjugated with Alexa 555 dye is added andleft to incubate for 1 hour before being washed again with PBS. Thesubstrate is heat shrunk in an oven before imaged using a fluorescencemicroscope.

FIG. 2A-D show microscopic pictures of a textured surface of the film atindicated magnification levels.

FIG. 3 a illustrates the process of shrinking a surface carrying abiological sample.

FIG. 3 b includes images showing the detected signal before (upper) andafter (lower) shrinking.

FIG. 4 presents a bar chart showing the detection efficiency before(left) and after (right) shrinking.

FIG. 5 a-c include images showing the fluorescence intensity for (a) DNAhybridized on glass slide (b) single-stranded DNA (non-hybridized) onsilica polyolefin (PO) (c) DNA hybridized on silica PO.

FIG. 6 presents a bar chart showing the detection efficiency before(left) and after (right) shrinking as reflected in the images of FIG. 5.

DETAILED DESCRIPTION Definitions

As used herein, certain terms may have the following defined meanings

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination when used for the intendedpurpose. Thus, a composition consisting essentially of the elements asdefined herein would not exclude trace contaminants or inert carriers.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps for preparing the intendeddevice. Embodiments defined by each of these transition terms are withinthe scope of this invention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above.

A “thermoplastic material” is intended to mean a plastic material whichshrinks upon heating or upon release of prestress such as a stresscreated by stretching. In one aspect, the thermoplastic materials arethose which shrink uniformly without distortion. The shrinking can beeither bi-axially (isotropic) or uni-axial (anisotropic). Suitablethermoplastic materials for inclusion in the methods of this inventioninclude, for example, shape memory polymers, polyolefin, polyethylene,high molecular weight polymers such as acrylonitrile butadiene styrene(ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate(EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, includingFEP, PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVCalloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal),polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile),polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone(PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutyleneterephthalate (PBT), polyethylene terephthalate (PET), PolycyclohexyleneDimethylene Terephthalate (PCT), polycarbonate (PC),polyhydroxyalkanoates (PHAs), polyketone (PK), polyester polyethylene(PE), polyetheretherketone (PEEK), polyetherimide (PEI),polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC),polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP),polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide(PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU),polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) and spectralon.

In some aspects, the thermoplastic material encompasses polyolefin. Apolyolefin is a polymer produced from a simple olefin (also called analkene) as a monomer. For example, polyethylene is the polyolefinproduced by polymerizing the olefin ethylene. Polypropylene is anothercommon polyolefin which is made from the olefin propylene.

In some aspects, the thermoplastic material encompasses shape memorypolymers (SMPs). SMPs are polymeric smart materials that have theability to return from a deformed state (temporary shape) to theiroriginal (permanent) shape induced by an external stimulus (trigger),such as temperature change.

Commercially available thermoplastic materials include, withoutlimitation, “Shrinky-Dink” and Solupore®. Shrinky-Dink is a commercialthermoplastic which is used a children's toy. Solupore® is availablefrom Lydall, Inc. of Manchester, Conn.

As used herein, “labels” or “detectable labels” are chemical orbiochemical moieties useful for labeling a nucleic acid. “Labels”include fluorescent agents, chemiluminescent agents, chromogenic agents,quenching agents, radionucleotides, enzymes, substrates, cofactors,inhibitors, nanoparticles, magnetic particles, and other moieties knownin the art. Labels are capable of generating a measurable signal and maybe covalently or noncovalently joined to an oligonucleotide ornucleotide.

In illustrative embodiments, the antibodies may be labeled with a“fluorescent dye” or a “fluorophore.” As used herein, a “fluorescentdye” or a “fluorophore” is a chemical group that can be excited by lightto emit fluorescence. Some fluorophores may be excited by light to emitphosphorescence. Dyes may include acceptor dyes that are capable ofquenching a fluorescent signal from a fluorescent donor dye. Dyes thatmay be used in the disclosed methods include, but are not limited to,the following dyes and/or dyes sold under the following trade names: 1,5IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (HAT);5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; AlizarinComplexon; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; AMCA(Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC(Allophycocyanin); APC-Cy7; APTS; Astrazon Brilliant Red 4G; AstrazonOrange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); Berberine Sulphate; Beta Lactamase; BFP blueshifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane;Bisbenzamide; Bisbenzimide (Hoechst); Blancophor FFG; Blancophor SV;BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570;Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X;Bodipy 665/676; Bodipy FL; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6GSE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR;Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; BrilliantSulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green;Calcium Orange; Calcofluor White; Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP—Cyan Fluorescent Protein;CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH);CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h;Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; CoelenterazineO; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTCFormazan; Cy2™; Cy3. 1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™;Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; DansylAmine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride;DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP);Dichlorodihydrofluorescein Diacetate (DCFH); DiD—Lipophilic Tracer; DiD(DiIC18(5)); DIDS; Dihydrorhodamine 123 (DHR); DiI (DiIC18(3));Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DNP; Dopamine;DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin;Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1(EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; FastBlue; FDA; Feulgen (Pararosaniline); Flazo Orange; Fluo-3; Fluo-4;Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM 4-46; FuraRed™; Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B;Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF;GeneBlazer (CCF2); GFP(S65T); GFP red shifted (rsGFP); GFP wild type,non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indo-1; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF;Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Nile Red; NED™; Nitrobenzoxadidole;Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant IavinEBG; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488;Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline(Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev;Phorwite RPA; Phosphine 3R; Phycoerythrin B [PE]; Phycoerythrin R [PE];PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3;PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI);PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2;Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G;Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; RhodamineBG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine;Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine;R-phycoerythrin (PE); RsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI;Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; SevronBrilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (superglow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline); SITS(Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARFcalcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen;SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;TET™; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; TexasRed-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; ThiazoleOrange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; ThiozoleOrange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3;TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; VIC®; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange;Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3;and salts thereof.

Fluorescent dyes or fluorophores may include derivatives that have beenmodified to facilitate conjugation to another reactive molecule. Assuch, fluorescent dyes or fluorophores may include amine-reactivederivatives such as isothiocyanate derivatives and/or succinimidyl esterderivatives of the fluorophore.

Methods for Preparing a Sample for Analysis

One embodiment of the present disclosure provides a method for preparinga sample for detection, comprising shrinking a thermoplastic materialcomprising a sample disposed on the thermoplastic material, therebyconcentrating the sample on the thermoplastic material.

Likewise, another embodiment provides a method for detecting a samplecomprising (A) disposing the sample on a thermoplastic material, (B)shrinking the thermoplastic material, and (C) detecting the sample.

In one aspect, the sample comprises a detectable label. In anotheraspect, the detectable label comprises a fluorescent agent. In yetanother aspect, the sample is a biological sample.

The thermoplastic material can be pre-stressed prior to shrinking. Insuch a case, the shrinking can be achieved by removing the stress. Sucha stress can simply be stretching, either uniaxially or biaxially.

Alternatively, the shrinking can be achieved by heating the material.Depending on the material and desired scale of texture, the temperaturecan vary. In one aspect, the heating is at least about 200° F., or atleast about 250° F., or at least about 275° F., or at least about 300°F., or at least about 350° F.

In some aspects, the material is treated with a plasma before theshrinking. It has been demonstrated that plasma treatment of athermoplastic material, such as a polyethylene (PE) film, creates astiff layer at the surface of a relatively softer bulk PE. Leveragingthe inherent refraction properties of the thermoplastic material atelevated temperature, the mismatch in stiffness between two layers willcause the stiff outer layer to buckle and form controllable textures orwrinkles.

Plasmas can be prepared with methods known in the art and can varydepending on availability of sources. In one embodiment, the plasma isoxygen plasma, helium plasma, or hydrogen plasma. In a particularembodiment, the plasma is oxygen plasma.

The duration of plasma treatment can vary and depend on the desiredscale of the texture and/or the thermoplastic material, for instance. Inone aspect, the plasma treatment takes more than about 10 seconds, oralternatively more than about 20 seconds, about 30 seconds, about 1minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5minutes, about 7 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 25 minutes, or about 30 minutes. In another aspect, theplasma treatment takes less than about 60 minutes, or alternatively lessthan about 45 minutes, about 30 minutes, about 25 minutes, about 20minutes, about 15 minutes, about 10 minutes, about 7 minutes, about 5minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1minute, about 30 seconds, or about 20 seconds. In some aspects, thetreatment is carried out in a closed chamber. In some aspects, thetreatment is carried out in a handheld corona discharger.

The thermoplastic material can be pre-stressed prior to the plasmatreatment. In such a case, the shrinking can be achieved by removing thestress. Such a stress can simply be stretching, either uniaxially orbiaxially.

Alternatively, the shrinking can be achieved by heating the material.Depending on the material and desired scale of texture, the temperaturecan vary. In one aspect, the heating is at least about 200° F., or atleast about 250° F., or at least about 275° F., or at least about 300°F., or at least about 350° F.

Shrinking of the material can be uniaxial or biaxial. When the materialis shrunk uniaxially, the texture may be one dimensional. When thematerial is shrunk biaxially, the texture may be two dimensional.

In some embodiments, the material is shrunk, uniaxially or biaxially, byat least about 60%, or alternatively at least about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99% from itsoriginal size.

Still, in some aspects, the material is etched, prior to deposition ofthe sample, to provide room for holding the sample. Such etching, in oneembodiment, also contributes to the enhanced signal detection. Methodsof etching and related shrinking are provided in WO 2009/064816, thecontent of which is incorporated into the present disclosure in itsentirety, by reference.

In one aspect, the sample is attached to the material. The attachmentcan be covalent or non-covalent. In one aspect, the attaching iscovalent. In a particular aspect, the covalent attachment involves alinker, such as but not limited to, biotin and Poly (L-glutamic acid)(Pglu). In one aspect, the sample is a biological sample, including butnot limited to protein, nucleic acids, such as DNA and RNA.

Shrinking of the material can be uniaxial or biaxial. When the materialis shrunk uniaxially, the texture may be one dimensional. When thematerial is shrunk biaxially, the texture may be two dimensional.

In some embodiments, the material is shrunk, uniaxially or biaxially, byat least about 60%, or alternatively at least about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99% from itsoriginal size.

In some embodiments, the concentration of the sample is increased by atleast about 5 fold, or alternatively about 5, or about 6, or about 7, orabout 8, or about 9, or about 10, or about 12, or about 14, or about 16,or about 18, or about 20, or about 25, or about 30, or about 40, orabout 50, or about 60, or about 70, or about 80, or about 90, or about100 or about 200 fold.

Further, some embodiments of the present disclosure provide samplesprepared by any of the disclosed methods.

EXPERIMENTAL EXAMPLES Example 1

This example illustrates preparation of an antibody forfluoroimmunoassay. A thin layer of silica is deposited on a heat shrinkfilm, polyolefin, by an E-beam evaporation process. Biologicalmolecules, in this case rabbit immunoglobin (IgG) is microcontactprinted on the solid substrate and then blocked with blocking solutionfor 2 hours at room temperature. After 2 hours, the blocking solution iswashed and then the goat anti-rabbit conjugated to AlexaFluor 555 isadded and after 2 hr incubation the fluorescence signal is measured.After measuring the fluorescence signal, the sample is shrunk in an ovenby heating sequentially to 115° C. for 30 minutes, 135° C. for 10minutes, and 155° C. The inherent ability of the polyolefin polymer toshrink helps with increasing the concentration of the protein andincreasing the fluorescent signal enhancement by 16 fold. Theexperimental procedure is illustrated in FIG. 1. FIG. 2A-D show thenanostructure of the shrunk surface of the film.

Bodily fluids such as saliva which have antigens that can be recognizedby the primary antibody can also be assayed the same way.

Example 2

This example demonstrates that amplification in sample signal can beachieved by shrinking the thermoplastic surface. The amplificationutilizes a protein's ability to naturally adsorb onto a surface and thesubsequent increase in detection signal is attributed to concentratingthe sample on the thermoplastic material and increasing the surfacearea. Further, the method illustrated here involves chemicallyfunctionalizing the thermoplastic material for covalent attachment ofbiological samples.

Modification of the clean thermoplastic surface starts with depositing athin layer of silica onto the surface by ion beam sputter coater,followed by oxygen plasma treatment for the introduction of hydroxylgroups. The surface is then aminated by submerging into a 5% (v/v)(3-aminopropyl)trimethoxysilane (APTMS) ethanolic solution for 2 hoursat room temperature. The thermoplastic substrate is then washed with100% ethanol and ddH₂O, and allowed to cure overnight at roomtemperature. The aminated thermoplastic surface is reacted withNHS-ester activated biotin (1 mg/ml) for 2 hrs, washed twice with 1×PBSand ddh₂O, and then incubated with 1 μL volumes of streptavidin-TRITCfor 2 hrs (10 μg/ml). Fluorescent signal is measured. The thermoplasticsubstrates are shrunk by heating to T=160° C. and fluorescent signal ismeasured again. This procedure is illustrated in FIG. 3 a.

The observed fluorescent signal of the shrunk thermoplastic substratesdemonstrates signal enhancement ranging between 55 to 75 fold (seeactual images in FIG. 3 b and comparison charts in FIG. 4).

Example 3

This is another example demonstrating that amplification in samplesignal can be achieved by shrinking the thermoplastic surface.

This procedure involves fabricating a DNA microarray by laser etching amicrowell pattern in polyolefin (PO) sealing tape and adhering to apolystyrene (PS) or PO thermoplastic. Features are etched into theexposed thermoplastic surface and silica is sputtered on top. Isopropylalcohol (IPA) is used to remove the PO sealing tape without disturbingthe silica. The exposed silica-coated wells are treated with oxygenplasma to introduce hydroxyl groups and then chemically modified with 5%(v/v) (3-aminopropyl)trimethoxysilane (APTMS) ethanolic solution for 4hours at room temperature. Poly (L-glutamic acid) (Pglu) is then reactedwith the aminated thermoplastic surface in the presence of1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-Hydroxysuccinimide(NHS). Single stranded DNA is flowed over the substrate to bind to thePglu through the formation of amide bonds. The thermoplastic substrateis shrunk by heating to T=160° C. Complementary stranded DNA tagged withCy3 is flowed over the shrunk substrate and fluorescent signal ismeasured (FIG. 5).

The observed fluorescent signal of biological sample shows a signalenhancement of around 90 fold (FIGS. 5 and 6).

While the present invention is exemplified and illustrated by the use ofpolystyrene sheets to fabricate channel structures and molds, it wouldbe obvious to those of skill in the art that any thermoplastic receptivematerial that can be patterned to control the dimensions of the channeldefining walls and thereby their size, can be used to fabricate thedevices disclosed and claimed herein. In addition, although severalother embodiments of the invention are described herein in detail, itwill be understood by those skilled in the art that variations may bemade thereto without departing from the spirit of the invention or thescope of the appended claims.

1. A method for preparing a sample for detection, comprising shrinking athermoplastic material comprising a sample disposed on the thermoplasticmaterial, thereby concentrating the sample on the thermoplastic material2. The method of claim 1, wherein the sample comprises a detectablelabel.
 3. The method of claim 2, wherein the detectable label comprisesa fluorescent agent.
 4. The method of claim 1, wherein the material ispre-stressed prior to the shrinking and the shrinking comprises removingthe stress.
 5. The method of claim 1, wherein the shrinking comprisingheating the material.
 6. The method of claim 1, wherein the shrinking isuniaxial or biaxial.
 7. The method of claim 1, wherein the material isshrunk by at least 60%.
 8. The method of claim 1, wherein thethermoplastic material comprises a high molecular weight polymer,polyolefin, a shape memory polymer, polyethylene, acrylonitrilebutadiene styrene (ABS), acrylic, celluloid, cellulose acetate,ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL),fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), ionomerskydex, a trademarked acrylic/PVC alloy, liquid crystal polymer (LCP),polyacetal (POM or Acetal), polyacrylates (Acrylic), polyacrylonitrile(PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI),polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene(PB), polybutylene terephthalate (PBT), polyethylene terephthalate(PET), Polycyclohexylene Dimethylene Terephthalate (PCT), polycarbonate(PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyesterpolyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI),polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC),polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP),polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide(PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU),polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) or spectralon.9. The method of claim 8, wherein the thermoplastic material comprisespolyolefin.
 10. The method of claim 8, wherein the thermoplasticmaterial comprises polyethylene.
 11. The method of claim 1, wherein thesample is attached to the material.
 12. The method of claim 11, whereinthe sample if covalently attached to the material.
 13. The method ofclaim 1, wherein the sample comprises a biological sample.
 14. Themethod of claim 1, wherein the sample comprises a protein.
 15. Themethod of claim 1, wherein the sample comprises a polynucleotide. 16.The method of claim 1, wherein the material is treated with a plasmabefore the shrinking.
 17. The method of claim 16, wherein the plasma isoxygen plasma.
 18. The method of claim 1, wherein the material is etchedbefore the sample is deposited on the material.
 19. A sample prepared bya method of claim
 1. 20. A method for detecting a sample comprising (A)disposing the sample on a thermoplastic material, (B) shrinking thethermoplastic material, and (C) detecting the sample.